I am at a oligonucleotide facility at UNC. We synthesize approximately
500 oligos a month. We have been following the discussion about dG-CE
phosphoramidites falling out of solution. By using the following
procedures we are able to keep our phosphoramidites on the machine for as
long as six weeks for A, C, and T, and four weeks for G. These times are
some what shorter in the summer time due to the fact that the relative
humidity around here reaches 99.44 %
Dilute your phosphoramidites by hand. Don't use the "auto dilute"
procedures on your machine. They never mix the solution sufficiently.
When diluting by hand use a small bottle of acetonitrile, 100 ml or less,
preferable one with a septum. These are available from several reagent
suppliers but not ABI. Use clean glass syringes that you keep in an
oven. Have a separate syringe for each base, and allow the syringe to
cool before you use it. If a syringe has been sitting out of the oven for
more than 30 min. to an hour put it back in for a few minutes. Use a
small disposable vent needle in the septum to allow you to dilute the
phosphoramidite. Once you have diluted the phosphoramidite, vortex it
very well and inspect it with a flashlight to make sure that there are no
undissolved particles. If you don't use a complete bottle of diluent at
one time you must blanket the top of the acetonitrile with argon. To do
this, use two disposable needles. Place one of the needles on one of your
synthesizers column positions and poke it through the septum on the
diluent bottle. Then use manual control to do a reverse argon flush(On
ABI synthesizers) Use the second needle to vent the pressure while doing
the argon flush. The argon is heavier than air and will push it out the
vent needle. After about 20 seconds take out the vent needle and continue
to pressurize for 20 more seconds. This keeps air from seeping back into
the bottle. Wrap the top of the bottle with parafilm and store in a
dessicator. All of this seemingly anal nonsense does two things. First,
it makes sure the phosphoramidites are well mixed, something that is
impossible with the auto dilute procedures. Second, and more importantly,
this keeps the acetonitrile diluent extremely dry. Not only does water in
the acetonitrile affect the coupling reaction, but also the solubility of
the phosphoramidites.
Other things you can do to keep your phosphoramidites in solution and your
synthesizers free of blockages.
1) Store bottles of diluent and undiluted phosphoramidites in a dessicator
2) Be sure to perform the begin procedure before synthesis if the
synthesizer hasn't been run for more than 4 hours.
3) Don't dilute the phosphoramidites until you need them.
4) If you are diluting RNA, labeling, or other special phosphoramidites,
use a disposable plastic syringe. DON'T REUSE these plastic syringes. The
rubber tips on the plungers are soluble in acetonitrile over time.
5) If you are synthesizing 40 or 50 nm oligos try double diluting the
phosphoramidites. This lower concentration is easier to keep in solution.
Please send any replies to my email address or post to this newsgroup.
Wade Turner
Nucleic Acids Core Facility
University of North Carolina at Chapel Hill
wwturner at med.unc.edu